Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes.
A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.
Irvine
J W
JW
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Roberts
S F
SF
Lindberg
I
I
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010450:Endopeptidases / Q000378:metabolism*; D004745:Enkephalins / Q000378:metabolism; D007610:Kallikreins / Q000097:blood / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008832:Microchemistry / Q000379:methods; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D012834:Silver; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity; D013462:Sulfur Radioisotopes; D014357:Trypsin / Q000378:metabolism
D004745:Enkephalins; D011498:Protein Precursors; D011506:Proteins; D013462:Sulfur Radioisotopes; C029992:proenkephalin; D012967:Sodium Dodecyl Sulfate; D012834:Silver; D008715:Methionine; D010450:Endopeptidases; D007610:Kallikreins; D012697:Serine Endopeptidases; C060973:membrane-bound adrenal trypsin-like enzyme; D002918:Chymotrypsin; D014357:Trypsin